H-NS oligomerization domain structure reveals the mechanism for high order self-association of the intact protein

H-NS plays a role in condensing DNA in the bacterial nucleoid. This 136 amino acid protein comprises two functional domains separated by a flexible linker. High order structures formed by the N-terminal oligomerization domain (residues 1-89) constitute the basis of a protein scaffold that binds DNA via the C-terminal domain. Deletion of residues 57-89 or 64-89 of the oligomerization domain precludes high order structure formation, yielding a discrete dimer. This dimerization event represents the initial event in the formation of high order structure. The dimers thus constitute the basic building block of the protein scaffold. The three-dimensional solution structure of one of these units (residues 1-57) has been determined. Activity of these structural units is demonstrated by a dominant negative effect on high order structure formation on addition to the full length protein. Truncated and site-directed mutant forms of the N-terminal domain of H-NS reveal how the dimeric unit self-associates in a head-to-tail manner and demonstrate the importance of secondary structure in this interaction to form high order structures. A model is presented for the structural basis for DNA packaging in bacterial cells.     

Role of the N-terminal domain of the human DMC1 protein in octamer formation and DNA binding

The DMC1 protein, a eukaryotic homologue of RecA that shares significant amino acid identity with RAD51, exhibits two oligomeric DNA binding forms, an octameric ring and a helical filament. In the crystal structure of the octameric ring form, the DMC1 N-terminal domain (1-81 amino acid residues) was highly flexible, with multiple conformations. On the other hand, the N-terminal domain of Rad51 makes specific interactions with the neighboring ATPase domain in the helical filament structure. To gain insights into the functional role of the N-terminal domain of DMC1, we prepared a deletion mutant, DMC1-(82-340), that lacks the N-terminal 81 amino acid residues from the human DMC1 protein. Analytical ultracentrifugation experiments revealed that, whereas full-length DMC1 forms a octamer, DMC1-(82-340) is a heptamer. Furthermore, DNA binding experiments showed that DMC1-(82-340) was completely defective in both single-stranded and double-stranded DNA binding activities. Therefore, the N-terminal domain of DMC1 is required for the formation of the octamer, which may support the proper DNA binding activity of the DMC1 protein.     

Crystal structure of human gamma-butyrobetaine hydroxylase

Gamma-butyrobetaine hydroxylase (GBBH) is a 2-ketoglutarate-dependent dioxygenase that catalyzes the biosynthesis of l-carnitine by hydroxylation of gamma-butyrobetaine (GBB). l-carnitine is required for the transport of long-chain fatty acids into mitochondria for generating metabolic energy. The only known synthetic inhibitor of GBBH is mildronate (3-(2,2,2-trimethylhydrazinium) propionate dihydrate), which is a non-hydroxylatable analog of GBB.To aid in the discovery of novel GBBH inhibitors by rational drug design, we have solved the three-dimensional structure of recombinant human GBBH at 2.0 Å resolution. The GBBH monomer consists of a catalytic double-stranded β-helix (DBSH) domain, which is found in all 2KG oxygenases, and a smaller N-terminal domain. Extensive interactions between two monomers confirm earlier observations that GBBH is dimeric in its biological state. Although many 2KG oxygenases are multimeric, the dimerization interface of GBBH is very different from that of related enzymes.The N-terminal domain of GBBH has a similar fold to the DUF971 superfamily, which consists of several short bacterial proteins with unknown function. The N-terminal domain has a bound Zn ion, which is coordinated by three cysteines and one histidine. Although several other 2KG oxygenases with known structures have more than one domain, none of them resemble the N-terminal domain of GBBH. The N-terminal domain may facilitate dimer formation, but its precise biological role remains to be discovered.The active site of the catalytic domain of GBBH is similar to that of other 2KG oxygenases, and Fe(II)-binding residues form a conserved His-X-Asp-X_n-His triad, which is found in all related enzymes.     

The phosphorylation domain of the 32-kDa subunit of replication protein A (RPA) modulates RPA-DNA interactions. Evidence for an intersubunit interaction

Replication protein A (RPA) is a heterotrimeric (subunits of 70, 32, and 14 kDa) single-stranded DNA-binding protein that is required for DNA replication, recombination, and repair. The 40-residue N-terminal domain of the 32-kDa subunit of RPA (RPA32) becomes phosphorylated during S-phase and after DNA damage. Recently it has been shown that phosphorylation or the addition of negative charges to this N-terminal phosphorylation domain modulates RPA-protein interactions and increases cell sensitivity to DNA damage. We found that addition of multiple negative charges to the N-terminal phosphorylation domain also caused a significant decrease in the ability of a mutant form of RPA to destabilize double-stranded (ds) DNA. Kinetic studies suggested that the addition of negative charges to the N-terminal phosphorylation domain caused defects in both complex formation (nucleation) and subsequent destabilization of dsDNA by RPA. We conclude that the N-terminal phosphorylation domain modulates RPA interactions with dsDNA. Similar changes in DNA interactions were observed with a mutant form of RPA in which the N-terminal domain of the 70-kDa subunit was deleted. This suggested a functional link between the N-terminal domains of the 70- and 32-kDa subunits of RPA. NMR experiments provided evidence for a direct interaction between the N-terminal domain of the 70-kDa subunit and the negatively charged N-terminal phosphorylation domain of RPA32. These findings suggest that phosphorylation causes a conformational change in the RPA complex that regulates RPA function.     

Sulerythrin,the smallest member of the rubrerythrin family,from a strictly aerobic and thermoacidophilic archaeon,Sulfolobus tokodaii strain 7

A protein corresponding to the N-terminal domain of rubrerythrin was isolated from a strictly aerobic archaeon, Sulfolobus tokodaii strain 7. The molecular mass was found to be 15.8 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 16278 Da by time-of-flight mass spectrometry and 34.5 kDa by gel filtration chromatography, suggesting that the protein is dimeric. Two mol iron and 1-2 mol zinc mol(-1) protein were detected. On addition of the azide ion, the absorption spectrum was greatly affected. The far UV circular dichroism spectrum suggested that the protein was mostly composed of alpha-helices. The N-terminal sequence completely matched the open reading frame, st2370, recently found on genome analysis of the organism. The protein was homologous to rubrerythrin but lacked a C-terminal rubredoxin domain. It was found in the genus Sulfolobus and therefore named sulerythrin; it is the smallest and first aerobic member of the rubrerythrin family.     

The N-terminal domain of the replication initiator protein RepE is a dimerization domain forming a stable dimer

The initiator protein RepE of the mini-F plasmid in Escherichia coli plays an essential role in DNA replication, which is regulated by the molecular chaperone-dependent oligomeric state (monomer or dimer). Crosslinking, ultracentrifugation, and gel filtration analyses showed that the solely expressed N-terminal domain (residues 1-144 or 1-152) exists in the dimeric state as in the wild-type RepE protein. This result indicates that the N-terminal domain functions as a dimerization domain of RepE and might be important for the interaction with the molecular chaperones. The N-terminal domain dimer has been crystallized in order to obtain structural insight into the regulation of the monomer/dimer conversion of RepE.     

Resolution of Holliday junction substrates by human topoisomerase I

Prompted by the close relationship between tyrosine recombinases and type IB topoisomerases we have investigated the ability of human topoisomerase I to resolve the typical intermediate of recombinase catalysis, the Holliday junction. We demonstrate that human topoisomerase I catalyzes unidirectional resolution of a synthetic Holliday junction substrate containing two preferred cleavage sites surrounded by DNA sequences supporting branch migration. Deleting part of the N-terminal domain (amino acid residues 1-202) did not affect topoisomerase I resolution activity, whereas a topoisomerase I variant lacking both the N-terminal domain and amino acid residues 660-688 of the linker domain was unable to resolve the Holliday junction substrate. The inability of the double deleted variant to mediate resolution correlated with the inability of this enzyme to introduce concomitant cleavage at the two preferred cleavage sites in a single Holliday junction substrate, which is a prerequisite for resolution. As determined by the gel electrophoretic mobility of native enzyme or enzyme crosslinked by disulfide bridging, the double deleted mutant existed almost entirely in a dimeric form. The impairment of this enzyme in performing double cleavages on the Holliday junction substrate may be explained by only one cleavage competent active site being formed at a time within the dimer. The assembly of only one active site within dimers is a well-known characteristic of the tyrosine recombinases. Hence, the obtained results may suggest a recombinase-like active site assembly of the double deleted topoisomerase I variant. Taken together the presented results consolidate the relationship between type IB topoisomerases and tyrosine recombinases.     

Role of the purine repressor hinge sequence in repressor function.

A protease-hypersensitive hinge sequence in Escherichia coli purine repressor (PurR) connects an N-terminal DNA-binding domain with a contiguous corepressor-binding domain. Binding of one molecule of dimeric repressor to operator DNA protects the hinge against proteolytic cleavage. Mutations in the hinge region impair repressor function in vivo. Several nonfunctional hinge mutants were defective in low-affinity binding to operator DNA in the absence of corepressor as well as in high-affinity corepressor-dependent binding to operator DNA, although binding of corepressor was similar to binding of the wild-type repressor. These results establish a role for the hinge region in operator binding and lead to a proposal for two routes to form the holoPurR-operator complex.     

Solution structure and biophysical properties of MqsA, a Zn-containing antitoxin from Escherichia coli

The gene ygiT (mqsA) of Escherichia coli encodes MqsA, the antitoxin of the motility quorum sensing regulator (MqsR). Both proteins are considered to form a DNA binding complex and to be involved in the formation of biofilms and persisters. We have determined the three-dimensional solution structure of MqsA by high-resolution NMR. The protein comprises a well-defined N-terminal domain with a Zn finger motif usually found in eukaryotes, and a defined C-terminal domain with a typical prokaryotic DNA binding helix-turn-helix motif. The two well-defined domains of MqsA have almost identical structure in solution and in the two published crystal structures of dimeric MqsA bound to either MqsR or DNA. However, the connection of the two domains with a flexible linker yields a large variety of possible conformations in solution, which is not reflected in the crystal structures. MqsA binds Zn with all four cysteines, a stoichiometry of 1:1 and a femtomolar affinity (K(a)≥10(17)M(-1) at 23°C, pH 7.0).     

Oligomeric state of the N-terminal domain of DnaT for replication restart in Escherichia coli

DNA replication stops when chemical or physical damage occurs to the DNA. Repairing genomic DNA and reloading the replication helicase are crucial steps for restarting DNA replication. The Escherichia coli primosome is a complex of proteins and DNA responsible for reloading the replication helicase DnaB. DnaT, a protein found in the primosome complex, contains two functional domains. The C-terminal domain (89–179) forms an oligomeric complex with single-stranded DNA. Although the N-terminal domain (1–88) forms an oligomer, the specific residues responsible for this oligomeric structure have not yet been identified.In this study, we proposed that the N-terminal domain of DnaT has a dimeric antitoxin structure based on its primary sequence. Based on the proposed model, we confirmed the site of oligomerization in the N-terminal domain of DnaT through site-directed mutagenesis. The molecular masses and thermodynamic stabilities of the site-directed mutants located at the dimer interface, namely Phe42, Tyr43, Leu50, Leu53, and Leu54, were found to be lower than those of the wild-type. Moreover, we observed a decrease in the molecular masses of the V10S and F35S mutants compared to the wild-type DnaT. NMR analysis of the V10S mutant revealed that the secondary structure of the N-terminal domain of DnaT was consistent with the proposed model. Additionally, we have demonstrated that the stability of the oligomer formed by the N-terminal domain of DnaT is crucial for its function. Based on these findings, we propose that the DnaT oligomer plays a role in replication restart in Escherichia coli.     

Flexible linkers leash the substrate binding domain of SspB to a peptide module that stabilizes delivery complexes with the AAA+ ClpXP protease

SspB dimers bind proteins bearing the ssrA-degradation tag and stimulate their degradation by the ClpXP protease. Here, E. coli SspB is shown to contain a dimeric substrate binding domain of 110-120 N-terminal residues, which binds ssrA-tagged substrates but does not stimulate their degradation. The C-terminal 40-50 residues of SspB are unstructured but are required for SspB to form substrate-delivery complexes with ClpXP. A synthetic peptide containing the 10 C-terminal residues of SspB binds ClpX, stimulates its ATPase activity, and prevents SspB-mediated delivery of GFP-ssrA for ClpXP degradation. This tripartite structure--an ssrA-tag binding and dimerization domain, a flexible linker, and a short peptide module that docks with ClpX--allows SspB to deliver tagged substrates to ClpXP without interfering with their denaturation or degradation.     

Generation of the BfiI restriction endonuclease from the fusion of a DNA recognition domain to a non-specific nuclease from the phospholipase D superfamily

The BfiI endonuclease cleaves DNA at fixed positions downstream of an asymmetric sequence. Unlike other restriction enzymes, it functions without metal ions. The N-terminal half of BfiI is similar to Nuc, an EDTA-resistant nuclease from Salmonella typhimurium that belongs to the phosphoplipase D superfamily. Nuc is a dimer with one active site at its subunit interface, as is BfiI, but it cuts DNA non-specifically. BfiI was cleaved by thermolysin into an N-terminal domain, which forms a dimer with non-specific nuclease activity, and a C-terminal domain, which lacks catalytic activity but binds specifically to the recognition sequence as a monomer. On denaturation with guanidinium, BfiI underwent two unfolding transitions: one at a relatively low concentration of guanidinium, to a dimeric non-specific nuclease; a second at a higher concentration, to an inactive monomer. The isolated C-terminal domain unfolded at the first (relatively low) concentration, the isolated N-terminal at the second. Hence, BfiI consists of two physically separate domains, with catalytic and dimerisation functions in the N terminus and DNA recognition functions in the C terminus. It is the first example of a restriction enzyme generated by the evolutionary fusion of a DNA recognition domain to a phosphodiesterase from the phospholipase D superfamily. BfiI may consist of three structural units: a stable central core with the active site, made from two copies of the N-terminal domain, flanked by relatively unstable C-terminal domains, that each bind a copy of the recognition sequence.     

The transposable element En/Spm-encoded TNPA protein contains a DNA binding and a dimerization domain

The En/Spm-encoded TNPA protein binds to 12-bp DNA sequence motifs that are present in the sub-termini of the transposable element. DNA binding of TNPA to monomeric and dimeric forms of the binding motif was analyzed by gel retardation and cross-linking studies. A DNA binding domain at the N-terminal and a dimerization domain at the C-terminal portion of TNPA were localized using deletion derivatives of TNPA. These domains are novel since no apparent homology has been found in the data bases. The stoichiometry of the TNPA-DNA complexes was analyzed. A special complex is formed with a tail-to-tail dimeric DNA binding motif, most probably involving two DNA-bound TNPA molecules that interact via their dimerization domains. In redox reactions the requirement for one or two disulfide bonds for DNA binding of TNPA was shown. The implications of these findings for the excision mechanism of En/Spm are discussed.     

Proteins of the kidney microvillar membrane. The amphipathic form of dipeptidyl peptidase IV.

Dipeptidyl peptidase IV was solubilized from the microvillar membrane of pig kidney by Triton X-100. The purified enzyme was homogeneous on polyacrylamide-gel electrophoresis and ultracentrifugation, although immunoelectrophoresis indicated that amino-peptidase M was a minor contaminant. A comparison of the detergent-solubilized and proteinase (autolysis)-solubilized forms of the enzyme was undertaken to elucidate the structure and function of the hydrophobic domain that serves to anchor the protein to the membrane. No differences in catalytic properties, nor in sensitivity to inhibition by di-isopropyl phosphorofluoridate were found. On the other hand, several structural differences could be demonstrated. Both forms were about 130,000 subunit mol.wt., but the detergent form appeared to be larger by no more than about 4,000. Electron microscopy showed both forms to be dimers, and gel filtration revealed a difference in the dimeric mol.wt. of about 38 000, mainly attributable to detergent molecules bound to the hydrophobic domain. Papain converted the detergent form into a hydrophilic form that could not be distinguished in properties from the autolysis form. A hydrophobic peptide of about 3500 mol.wt. was identified as a product of papain treatment. The detergent and proteinase forms differed in primary structure. Partial N-terminal amino acid sequences were shown to be different, and the pattern of release of amino acids from the C-terminus by carboxypeptidase Y was essentially similar. The results are consistent with a model in which the protein is anchored to the microvillar membrane by a small hydrophobic domain located within the N-terminal amino acid sequence of the polypeptide chain. The significance of these results in relation to biosynthesis of the enzyme and assembly in the membrane is discussed.